PCR does not normally use more than one pair of primers to amplify the same DNA region. This is because the DNA polymerase used in normal PCR contains 5′ to 3′ exonuclease activity. Therefore, if a reaction contained nested primers for the same strand of a target sequence, the inner extension strand would be degraded due to exonuclease activity of the polymerase. Conventional PCR containing multiple nested primers would therefore not increase PCR efficiency or sensitivity. The hydrolysis probe-based qPCR relies on the 5′ to 3′ exonuclease activity of Taq DNA polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection. To compare PCDR and hydrolysis probe-based qPCR, we performed a four-primer PCDR assay and two-primer hydrolysis probe (TaqMan) PCR assay. We used TaqMan Master Mix from Applied Biosystems; this contains AmpliTaq Gold DNA polymerase, which has 5′ to 3′ exonuclease activity. qPCR was carried out using a DENV3 detection probe with four of the pan-dengue primers and PCDR master mix or two of the primers and the TaqMan Master Mix. We first tested the four different combinations of primer pairs from two forward primers and two reverse primers in the hydrolysis probe-based qPCRs. As expected, the innermost pair of primers worked best (data not shown) and was used for comparison with PCDR. The sensitivity of PCDR and hydrolysis probe-based qPCR was determined using 10-fold dilutions of the DENV3 template DNA (Table 3). Representative amplification curves for 20 copies of template DNA in PCDR and hydrolysis probe-based qPCR are shown in Figure 4. The PCDR assays achieved consistently lower Cq values than the hydrolysis probe-based qPCR at all dilutions. At the lowest amount of 20 copies, the Cq value was 29.82 for PCDR using four primers compared with 33.91 for the hydrolysis probe-based qPCR.

To test whether PCDR improved the sensitivity of detecting actual dengue viral samples, RexD Aedes aegypti mosquitoes were infected with DENV3 virus by intrathoracic injection, and infection levels were confirmed by immunofluorescence assay on head squashes. RNA was extracted and converted to cDNA. The qPCRs were performed on undiluted and serially diluted cDNA (Table 4). PCDR showed increased efficiency over both two-primer assays, with improved Cq values, particularly for the reactions with a lower template concentration. Sequencing of the PCDR amplicons from these mosquito samples showed that no off-target amplicons were produced (data not shown).

Increased sensitivity of PCRs has been achieved previously by using nested primers. For example, nested PCR has been incorporated into quantitative assays for the detection of Mycobacterium tuberculosis (25); however two separate rounds of PCR are required, which increases the run time and the risk of cross-contamination.

In this report, we describe PCDR, a novel technique that increases the sensitivity of amplification, similar to the use of nested primers, but in a single, closed-tube reaction. This decreases the chance of contamination and reduces the run time to equivalent to or even less than normal qPCR, while using conventional qPCR platforms. In these proof-of-principle experiments, we were able successfully to amplify and detect dengue virus sequences from various sources and types of material; however the performance of PCDR in other contexts remains to be assessed.

The increased sensitivity obtained by PCDR would be useful in assays with low template DNA, for example in viral diagnostics. Using multiple primers here would also have an advantage over conventional PCR for detecting all variants, helping to overcome the problem of template variation due to the high mutation rate of many viruses. False negatives in viral diagnostics have already been reported due to mutations in the primer/probe hybridization sites (26), and diagnostic kits should ideally be able to cope with these changes without compromising on specificity. However, in this context, PCDR only uses a single probe, and therefore this method could also be prone to false negatives due to mutations in the probe-hybridization site. PCDR could also be utilized in multiplex format to determine different serotypes of the same species, in which the probes are designed to specifically bind to each serotype.

Acknowledgments

The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under the grant agreement 282589 and was supported by funds from the Regents of the University of California from The Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative. We are grateful to Dr. Angelo Scibetta for his review of the manuscript and helpful suggestions.

Competing interests

G.F., C.H., and L.A. are employees of Oxitec Ltd; G.F. and L.A. have equity interest in Oxitec Ltd. Oxitec, and GeneFirst have patents or patent applications related to the subject matter of this paper. G.F. and L.A. have equity interest in GeneFirst Ltd., which is developing molecular diagnostics methods and products.